The isolation of biomolecules, such as proteins and peptides, has become of an increased interest during the past years. Some biomolecules need to be isolated as a last step of a biotechnological method for the production thereof, for example in the preparation of protein-based pharmaceutical compounds. Similarly there is also a need to separate biomolecules for analytical purposes in order to be able to quantitate and identify the proteins and/or peptides present in a sample. Electrophoretic methods are commonly used in the separation step. A wide variety of methods are used for the detection and quantification of the separated proteins. For identification and characterisation of separated proteins MS methods are normally used as these methods are fast and require very small amounts of proteins and/or peptides.
In general terms, electrophoresis involves the movement of charged particles or ions in an electric field. The driving force for the electrophoretic transport of an ion or a particle is the product of the effective charge of the particle and the potential gradient, and the frictional resistance of the medium balances this force. The transport of a particle or ion is characterised by the electrophoretic mobility m, which is defined as the distance d travelled in the time t by the particle under the influence of the potential gradient E (m=d/tE). The electrophoretic mobility of proteins and peptides depend on the pH and the ionic strength of the medium in which the separation is done and of this reason the conductivity is given by some type of buffer components, which also control the pH and ionic strength of the medium. The systems generated in electrophoresis are gravitationally unstable and require some type of stabilisation. This has been achieved in variety of ways: by the use of density gradients generated of an electrophoretically immobile solute like glycerol; by performing the separation in capillaries, the capillary'space generated between two glass plates or in any other space of capillary dimensions generated on a chip; by doing the separation in paper or cellulose powder; by using a variety of gel-forming substances like starch, agarose or polyacrylamide. Gels like starch and acrylamide, but also linear polymers present in the liquid media in capillary geometry's, decrease the electrophoretic mobilities of proteins and peptides, and reinforce the dependence of the electrophoretic mobilities on the molecular weights of the proteins and/or peptides through the ‘sieving’ effect introduced by the polymer chains present in the media. It is common to include components in the separation medium that improve the solubility of the proteins and peptides to be separated. Examples of components used are well-known uncharged detergents like Triton and 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), but also urea is a commonly used additive.
Based on how the pH and ionic strength is established along the separation distance, basically three different types of electrophoretic methods can be distinguished: The first type is zone electrophoresis, in which separation takes place in a medium of constant pH and ionic strength established with a conventional low molecular weight buffer present in the medium during the separation. In zone electrophoresis the sample is applied either at the cathodic or anodic end of the separation medium. To start the electrophoresis from a sample zone with a narrow width is in zone electrophoresis essential. A sharp narrow starting zone can be generated either by using the retardation resulting when the sample components enter a sieving media, with the aid of discontinuous buffer system or a combination of these two means. If the sample components to be separated are anions applied cathodic, the gel buffer in a discontinuous system will contain a buffering base and an anion with high electrophoretic mobility at the pH given by the gel buffer. Examples of commonly used anions are chloride, sulphate or acetate. The buffer in the cathodic electrode chamber normally contains the same weak base as the gel buffer and a partially negatively charged compound, which should have a lower electrophoretic mobility than the mobility of the sample components in the electrode chamber and/or in the eventual stacking gel at the pH generated in this gel and with the sieving effects of the gel taken into account. Examples of compounds used for the latter purpose are week acids as borate or amphoteric substances like glycine, tricine, alanine or HEPES titrated to pH values higher than the pI value of the compound. As a consequence of the arrangement, the sample components will be concentrated (stacked) into a narrow sharp zone localised at the boundary between the high mobility gel buffer anion for example chloride, and the low mobility anionic compound, for example glycine, originating from the electrode chamber. When this zone enter the separation gel the combined effects of an increased retardation of the sample components in the gel, and the increase of the mobility of the low mobility compound due to a pH shift, will result in that the electrophoretic mobility of the sample components become lower than the mobility of the anionic compound originating from the cathodic electrode chamber.
Sodium dodecyl sulphate (SDS)-electrophoresis is a variant of zone electrophoresis, which separates polypeptides according to their molecular weight. The SDS masks the charge of the proteins themselves and the formed anionic complexes have in free solution approximately identical electrophoretic mobilities independent of the size of the polypeptide. The molecular weight dependence is generated with the use of a sieving media, polyacrylamide gel is the media most commonly used for the purpose. A common and advantageous approach in connection with SDS electrophoresis is to utilise gradient gels containing varying concentrations of polyacrylamide where the a polyacrylamide concentration increase in the transport direction of the SDS-protein complexes from the sample application point towards the cathode. The mobilities of the protein will steadily decrease during the transport through the gel as a result of the variation of the sieving effect. SDS-protein complexes will remain stacked and move in narrow sharp zone localised at the boundary between the zone containing the gel buffer anion and the separation zone as long as the SDS-protein complex has a mobility higher than the weak acid present in the separation zone. As a consequence complexes corresponding to high molecular weight will destack already at low polyacrylamide concentration in an early state of the experiment. Low molecular weight complexes will remain stacked to close to the end of the experiment.
In zone electrophoresis the most common ways to establish the convectional stabilisation needed is either to use systems with capillary dimensions or to use some kind of gel. There exist a large variety of commercially available gels intended for different zone electrophoretic applications and normally designed to be used with a specific instrument. Most commonly, these gels are wet, ready to use, containing the buffer components and all other substances required for the specific application. However, dry gels are also available, which then are rehydrated prior to use in solutions containing the suitable components required for the use.
The second type of the electrophoretic methods is isoelectric focusing (IEF), in which separation take place in a stationary pH gradient that occupies the whole separation distance and is arranged so that the pH in the gradient increases from anode towards the cathode. While other alternatives also exist, the pH gradients required in isoelectric focusing are in practice generated in two different ways:                (a) with the aid of a solution of carrier ampholytes. With carrier ampholytes is understood a mixture, which contains a very large number of different amphoteric molecules. The demand on these amphoteric molecules are that each one should comprise a number of charged or chargeable groups resulting in a good buffer capacity at the isoelectric point of the amphoteric molecule and contribute with the conductivity required. The isoelectric points of the molecules in the ampholyte span a range of values, with a sufficient number of different isoelectric points among the molecules in the mixture to produce essentially a continuum of values of the isoelectric points. Thus, when a container is filled with a solution of a carrier ampholyte and a voltage is applied across the solution with an acid as the anolyte and a base as the catholyte, the individual ampholyte molecules arrange themselves in order of increasing isoelectric point from anode towards the cathode. A variety of synthetic carrier ampholytes are commercially available, such as Pharmalyte™, and Ampholine™ (all from Amersham Biosciences, Uppsala, Sweden). Carrier ampholyte generated gradients are not truly stationary, but show a slow drift and change of shape with time.        (b) with an immobilised pH gradient in which case the charged or chargeable groups generating the pH gradient is bound either to the wall of a capillary system or to the matrix when some kind of gel is used to get convection stabilisation. The immobilised charged or chargeable groups used are normally a limited number of carboxylic groups or amino groups with different pK-values distributed within or close to the pH gradient, which is to be generated. The concentration of the charged or chargeable groups is varied along the separation distance in a manner causing the pH at which the wall or the gel matrix has a zero net charge to increase from the anode to the cathode. A commercially available example of a system for generation of immobilised pH gradients is the Immobiline II system™ (Amersham Biosciences, Uppsala, Sweden), wherein a pH gradient covalently attached to a polyacrylamide gel is formed. Immobilised pH gradients are truly stationary and today they are normally used together with carrier ampholytes. In this combination the immobilised gradient determine the resulting pH gradient, while the carrier ampholytes contribute with conductivity.        
The width of application zone is not critical in isoelectric focusing. In principal the sample can be mixed in to the separation medium and at the start of the separations be present all along the separation distance, but for analytical applications the sample is normally applied close to either the anode or the cathode. To provide the convectional stabilisation, capillaries and different types of gels are used also in isoelectric focusing . Examples of wet gels ready to use are Ampholine PAGplate™ gels, which exist for a number of pH ranges pH3.5–9.5, pH 4.0–6.5, pH 5.5–8.5 and pH 4.0–5.0. Examples of dry gels are Clen Gel IEF™ and Immobiline Dry Plate™ gels. A special variant of the latter type is the Immobiline DryStrip™ gels, which are designed to be used as first dimension in two-dimensional electrophoresis.
Besides that isoelectric focusing is used together with the convectional stabilisation means described as generally useful in connection with electrophoretic separation methods, it can also be used in chamber equipments. This type of equipment contains a number of compartments separated by membranes, which allow electrophoretic transport of carrier ampholytes and proteins between the chambers, but block the flow of liquid. A commercially available equipment of this type is the Iso-Prime™ (Amersham Biosciences, Uppsala, Sweden). The membranes used could either be uncharged or alternatively contain an immobilised pH. If the latter is the case, the immobilised pH will differ between the membranes and increase from the anode towards the cathode. Isoelectric focusing in chamber equipment has been used as prefractionation tool prior to 2-D electrophoresis, but also as mean for the purification of specific proteins.
The third type of the electrophoretic methods is isotachophoresis, in which the separation takes place in a region of varying pH and/or ionic strength. This region normally occupies a fraction of the total separation distance and is transported in the electric field during the separation. The mobilities of the proteins and/or peptides to be separated varies in the region in a manner, which makes them focus at different positions within the region where their respective transport velocity agrees with the velocity with which the gradient is transported. The pH variation could either be step-wise, generated with a limited number of compounds or alternatively it could be a continuous gradient generated with carrier ampholytes. An important application which fall in the latter category is non-equilibrium pH gradient electrophoresis (NEPHGE), which represents an alternative to IEF for separation of basic proteins in the first dimension of 2-D electrophoresis.
After an electrophoretic separation there is frequently a need to identify and characterise separated proteins and/or peptides, something normally done with mass spectrometric techniques. Especially SDS electrophoresis, as independent method or as second dimension in 2-D electrophoresis, is commonly combined with a subsequent identification and characterisation with MS. The normally procedure in this context is to cut out a gel plug containing a protein to be identified. Wash out eventual stain and other components present in the gel plug, which might disturb subsequent steps. Dry the gel plug down and then rehydrate the plug in a buffered trypsin containing solution. Generated peptide fragments is then extracted, resulting solution concentrated, applied and dried down on a MALDI target together with the matrix required for energy absorption at the wave length of the used laser. Identification from the generated peptide mass fingerprint is done by a search and comparison in a protein sequence database. Alternatively generated peptides can be analysed with ESI-MS or with MS/MS techniques in order to get amino acid sequence information as well as information on post-translational modifications.
In nature the sulphur containing side-chain of cysteines appear either as thiol groups, as disulphides connecting two cysteines or in a variety of other bonds, like the S-heme bonds or the iron-sulphur bonds found in many proteins involved in the respiratory chain. In the reducing environment existing in the cytosol, cysteinyl groups are normally present as thiol groups, while secreted or cell surface proteins often contain inter- and/or intra-chain disulphide bonds. The role of these disulphide bonds is to stabilise the three dimensional structure of proteins and also to keep the amino acid chains generating a protein or peptide together. Prior to an electrophoretic separation, it is common to reduce all inter- and intra-chain disulphide bonds present in the sample components to thiol groups. Whether the thiol groups are present originally, or generated in a reduction step, they very frequently create problems in electrophoretic separation methods, when the separation takes place at neutral or basic pH values. This is due to the high reactivity of thiol groups in this pH range and the fact that the reactions involved influence the behaviour of the proteins or peptides in the electrophoretic separations. The effect of the reaction of thiol groups is most pronounced in isoelectric focusing and becomes very conspicuous in 2D electrophoresis, a technique normally used to separate very large number of proteins. In 2-D electrophoresis as routinely performed today, the first dimension is usually an isoelectric focusing based on charge and the second dimension is a size-based sodium dodecyl sulphate (SDS) step. The first dimension focusing is conventionally performed in a polyacrylamide gel in the presence of a reducing agent, the function of which is to prevent thiol groups of sample proteins to oxidise or otherwise react during the focusing. Two commonly used reducing agents are dithiothreitol (DTT) and dithioerythritol (DTE), which are weak acids having pKa's of 8.3 and 9.0, respectively. Accordingly, both DTT and DTE are negatively charged at high pH values. Thus, during focusing in a gel that comprises a pH gradient, these substances will be transported away from the basic part of the gradient to be accumulated in a pH region of about 7–7.5. Thus, at basic pH values (pH higher than or equal to about 7) sample proteins' thiol groups will not be protected at all by the reducing agent. As the thiol groups are charged in this pH region, reaction of thiol groups will change the isoelectric point of the protein or peptide. If the conditions used result in a fast transport of proteins to the isoelectric point and the reaction rate for the consumption of thiol groups is comparatively slow, the observable result of a 2-D electrophoretic experiment is that a protein containing n thiol groups will appear in up to (n+1) protein spots in the resulting 2-D map, were these spots are connected with a faint streak. This kind of result is normally achievable only when anodic sample application is used, the separation distances are short and the pH gradient is steep. The need for anodic application is connected with that the reaction of thiol compounds is pH dependent and that cathodic application results in a fast formation of —S—S— bridges between molecules and generation of large protein aggregates. If high resolution is required, larger separation distances and flatter pH gradients have to be used, which decrease the rate with which proteins are transported. This intensify the streaking and with increasing separation distances and decreasing slope of the pH gradient the connected spots will gradually be converted to continuous streaks. The appearance of artifactual protein spots and/or streaks complicates or even prevents any accurate interpretation of the results.
Furthermore, the problems described for isoelectric focusing will appear in all kinds of charge-dependent electrophoretic separations performed at pH values higher than 7 as soon as the separation medium contains components which can react with the cysteinyl groups. It has long been considered a problem that residual unreacted acrylamide can form covalent adducts to proteins in conventional zone electrophoresis. Traces of remaining catalyst can similarly be expected to react with proteins and in all type of separations made in equipments where the separation media is in contact with air, oxidation of cysteinyl groups is expected. Urea added to get an improved protein solubility is another chemical which when present can react with thiol groups. As zone electrophoretic and isotachophoretic separations normally take much shorter time than isoelectric focusing, the heavy streaking that can appear with the latter technique is not normally noted. The problem observed is instead that each thiol containing proteins will appear as a number of bands, where the number of bands and the relative intensity of the bands will vary between experiments depending on the degree of oxidation of thiol groups.
In SDS electrophoresis the starting point is a sample containing proteins in which all cysteinyl groups present have been reduced to thiols. This is normally achieved by heating a protein containing sample to 95° for 3–5 minutes in a solution containing an excess of SDS and reducing agent, where the reducing agent normally used are either mercaptoethanol, DTT or DTE. SDS electrophoresis is normally performed in a polyacrylamide gel and most commonly with the well known buffer system according to Laemmli (Laemmli U.K (1970) Nature (London) 277, page 680). With this buffer system, the gel originally contains a Tris-chloride buffer of pH 8.7. The reduced sample containing SDS and a large excess of reducing agent is added at the cathodic end of the gel. The cathodic electrode buffer contains Tris, glycine and SDS. When the glycine and SDS enter the gel and substitute the chloride ions, a pH of approx. 9.5 will be established within the gel. At this pH mercaptoethanol as well as DTT and DTE have higher electrophoretic mobilities than glycine. As a consequence the reducing agent will immediately collect and concentrate in a narrow zone found between the zone containing the chloride ions originally present in the gel and the glycine zone in which the separation will take place. The passage of thiols through the gel will eliminate some compounds capable of reacting with cysteinyl groups. Especially when low molecular weight proteins and peptides are to be separated use of an acid with higher mobility than glycine can be required and tricine is for the purpose the most common choice. Also when storage stable polyacrylamide gels are used (pH of gel buffer <8) it is common to use acids with higher mobility than glycine. A number of different chemicals have been suggested and used in this context, examples besides tricine, are taurine and HEPES. Common to these compounds are, that in the pH range where they are used, their mobilities will be higher than the mobility of the thiol used as the reducing agent in the sample. Depending on the thiol used, some, or all the proteins and peptides to be separated, will move faster through the gel than the reducing agent. Irrespective if SDS-electrophoresis is run with glycine or a faster ion in the separation zone, the situation will be similar to the situation in isoelectric focusing at high pH values in the sense that there is no reducing agent present during the separation phase to protect the cysteinyl groups against reaction. The difference is that in SDS-electrophoresis the separation is independent of the charge of the protein. The described and known reactions of thiol groups in connection with SDS-electrophoresis are addition of acrylamide to form cysteinyl—S—β-propionamide and oxidation of thiol groups to form either inter- or intra-chain —S—S— bridges. Addition of one or a couple of acrylamide molecules to a protein marginally increases the mass of the protein, but has normally no detectable effect on the electrophoretic mobility of the protein-SDS complex in a polyacrylamide gel. Formation of inter-chain disulphide bridges, normally resulting in the generation of a dimer of a protein or peptide, result in drastic change in molecular weight and the electrophoretic mobility. Oxidation resulting in intra-chain disulphide bridges influences the shape of the SDS-protein complexes and result in small but detectable increase in the electrophoretic mobility. An oxidation product with deviating electrophoretic mobility will only be detected as a separated entity provided sufficient amount has been produced prior to that the product destacks. With a homogenous separation gel, which is a gel with constant acrylamide concentration, extra bands will only result if extensive oxidation takes place already in the stacking gel. Oxidation within the separation gel will only contribute with a diffuse background within the sample tracks. The situation becomes different in gradient gels especially for low molecular weight proteins containing at least two cysteines. In gradient gels it is normal to detect an extra artificial band or protein spot for this type of proteins. Even if the negative visible effects are less dramatic in SDS electrophoresis than in charge dependent electrophoretic separation methods, a solution to this problem is still desirable.
In connection with MS used for the identification and characterisation an initial step is digestion with trypsin. This step can not be run under reducing conditions as thiols like mercaptoethanol, DTT or DTE deactivate trypsin. None of the following steps involving extraction, concentration of generated peptides and drying down on the MALDI target together with matrix is done under reducing conditions. If reduced protein samples, where the cysteinyl groups are expected to be present as —SH, are digested with trypsin and used for identification with MALDI-TOF MS generated mass fingerprints, no masses corresponding to cystein containing peptides are found in the mass spectra. The probable reason is oxidation of the thiol resulting in —S—S— bridges, and maybe also a variety of other reactions, resulting in products with different masses negatively contributing to the background in the MALDI spectrum. For proteins separated with SDS electrophoresis prior to identification, cystein-containing peptides are detected as their —S-β-propionamide derivatives. The conversion of the thiol is incomplete and it seems as only the most reactive thiol groups are converted to propionamide derivatives to an extent, which allows the detection of the corresponding peptide in an MS spectrum. Clearly the degree of conversion will depend on the acrylamide concentration in the gel, which normally is high in home-cast gel but, as a result of the poisonous nature of acrylamide, kept low in commercial products. Method description exist for the alkylation of proteins with acrylamide prior to sequencing with conventional Edman degradation, which in principal should be possible to use either prior to the electrophoretic separation or between the electrophoretic separation and the MS identification. However, in reality, the reaction with acrylamide either is incomplete or alternatively ends up with the reaction of other nucleophils present in the proteins.
An approach frequently used in connection with 2-D electrophoresis is to use two equilibration steps between the first dimension and second dimension SDS electrophoresis. In the first step eventual —S—S— bridges formed are reduced with DTT and in second step available —SH groups are reacted with iodoacetamide. Although the conditions used allow detection of a number of cystein-containing peptides as acetamide adducts, this reaction is with the condition used also incomplete and cystein-containing peptides are missing in the resulting mass spectra As with acrylamide, use of higher iodoacetamide concentrations and/or longer reaction times result in the reaction of other nucleophilic groups present in the protein.
Several solutions to avoid the above-described problems have been proposed. For example, phosphines, such as tributylphosphine and tris-hydroxypropyl phosphine have been used to replace DTT and DTE as reducing agents. However, the tested phosphines have shown to entail problems due to low solubility, and also to result in various undesired side effects. As an alternative, alkylation of the thiol groups of proteins before electrophoresis has been suggested. However, this approach has been shown to result sometimes in a non-complete alkylation, and sometimes in undesired side effects. Moreover, it has been suggested to allow the reducing agent to continuously leak into the gel from the cathode side of the apparatus. However, this method requires very careful attention to avoid a too large amount of reducing agent, which may cause problems, while avoiding adding too small an amount, which will yield an unsatisfactory reduction.
The problem with the disappearance of thiol containing peptides in connection with MS is a problem not solely connected to the separation of proteins with electrophoretic methods and the modifications of thiols resulting during this separation. Independent of how the protein is purified the steps prior to MS identification is reduction of the proteins with MeSH, DTT or DTE followed by an alkylation step in which the commonly used alkylating agents are iodoacetic acid, iodoacetamide, vinylpyridine or acrylamide. After alkylation and prior to trypsin digestion a desalting step is normally done, after which the sample together with matrix is applied to a MALDI-target and dried down. The alkylation step is introduced to allow the detection of cystein containing peptides in the resulting mass spectra. As already discussed this type of alkylation reaction is not ideal for the purpose. Either the result is only a partial conversion of the thiol groups present in the sample, alternatively other nucleophilic groups present in the sample will react. In the former case peaks corresponding to some cysteinyl-containing peptides will be missing in the resulting mass spectrum, in the latter case artefactual peaks with non-predictable masses will appear in the mass spectra. Thus, there is a need of a reaction with better selectivity, which allow a complete or close to complete conversion of the thiol groups without causing any side reaction, will clearly be advantageous prior to identification and characterisation of proteins and peptides with mass spectrophotometric methods.
Accordingly, there is a need in this field of a method, which eliminates the negative effects of reactions of thiol groups on separations, especially methods performed with electrophoretic methods. Simultaneously there is need for a method or reaction, which allow efficient and close to complete conversion of all cysteinyl groups, present in a protein and/or peptide containing sample, to a defined form possible to detect and study with mass spectrometric methods, at the same time as modifications of other groups present are avoided or at least kept to a minimum.